Identification of proteoglycans as the APRIL-specific binding partners.

B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

Presence of JC virus-specific CTL in the cerebrospinal fluid of PML patients: rationale for immune-based therapeutic strategies.

OBJECTIVES: There is urgent need of a treatment for progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). To evaluate the rationale for immunotherapy of PML, we explored whether JCV-specific cytotoxic T lymphocytes (CTL) can penetrate the central nervous system (CNS). In addition, we studied the breadth of their T-cell receptor (TCR) repertoire, and sought to establish a reliable method to expand these cells in vitro. DESIGN AND METHODS: We enrolled 18 patients in this study, including 16 with proven or possible PML (15 HIV-positive and one HIV-negative), and two HIV-positive patients with other neurological diseases. Detection of JCV-specific CTL in the blood and the cerebrospinal fluid was performed by Cr release and tetramer staining assays in 15 patients. RESULTS: Of 11 PML patients with analyzable cerebrospinal fluid (CSF), two had no detectable JCV-specific CTL in the blood and CSF and died 3.7 and 7.2 months later. The nine remaining patients had an inactive course of PML and detectable JCV-specific CTL in the blood. In addition, four of them (44%) also had detectable JCV-specific CTL in the CSF. Both HIV-positive patients with OND had detectable JCV-specific CTL in the blood and one in the CSF. Using tetramer technology, we obtained highly enriched JCV-specific CTL lines that were able to kill target cells presenting JCV peptides. The breadth of the TCR repertoire was CTL epitope dependent. CONCLUSIONS: These results indicate that JCV-specific CTL are present in the CNS of PML patients and pave the way for an immune-based therapeutic approach.

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

Melan-A-specific cytotoxic T cells are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4.

PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time.

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells.

ABSTRACT: BACKGROUND: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro. METHODS: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment. RESULTS: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA. CONCLUSIONS: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Genetic variability of the mTOR pathway and prostate cancer risk in the European Prospective Investigation on Cancer (EPIC)

The mTOR (mammalian target of rapamycin) signal transduction pathway integrates various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size. In addition, recent evidence has pointed to an interplay between the mTOR and p53 pathways. We investigated the genetic variability of 67 key genes in the mTOR pathway and in genes of the p53 pathway which interact with mTOR. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a study of 815 prostate cancer cases and 1,266 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC). We chose the SNPs (n = 11) with the strongest association with risk (p<0.01) and sought to replicate their association in an additional series of 838 prostate cancer cases and 943 controls from EPIC. In the joint analysis of first and second phase two SNPs of the PRKCI gene showed an association with risk of prostate cancer (ORallele = 0.85, 95% CI 0.78–0.94, p = 1.3×10−3 for rs546950 and ORallele = 0.84, 95% CI 0.76–0.93, p = 5.6×10−4 for rs4955720). We confirmed this in a meta-analysis using as replication set the data from the second phase of our study jointly with the first phase of the Cancer Genetic Markers of Susceptibility (CGEMS) project. In conclusion, we found an association with prostate cancer risk for two SNPs belonging to PRKCI, a gene which is frequently overexpressed in various neoplasms, including prostate cancer.

Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells.

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

In vitro attachment of glycosyl-inositolphospholipid anchor structures to mouse Thy-1 antigen and human decay-accelerating factor.

Glycosyl-inositolphospholipid (GPL) anchoring structures are incorporated into GPL-anchored proteins immediately posttranslationally in the rough endoplasmic reticulum, but the biochemical and cellular constituents involved in this "glypiation" process are unknown. To establish whether glypiation could be achieved in vitro, mRNAs generated by transcription of cDNAs encoding two GPL-anchored proteins, murine Thy-1 antigen and human decay-accelerating factor (DAF), and a conventionally anchored control protein, polymeric-immunoglobulin receptor (IgR), were translated in a rabbit reticulocyte lysate. Upon addition of dog pancreatic rough microsomes, nascent polypeptides generated from the three mRNAs translocated into vesicles. Dispersal of the vesicles with Triton X-114 detergent and incubation of the hydrophobic phase with phosphatidylinositol-specific phospholipases C and D, enzymes specific for GPL-anchor structures, released Thy-1 and DAF but not IgR protein into the aqueous phase. The selective incorporation of phospholipase-sensitive anchoring moieties into Thy-1 and DAF but not IgR translation products during in vitro translocation indicates that rough microsomes are able to support and regulate glypiation.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

MAGE-A3 and MAGE-A4 specific CD4(+) T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes.

Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from healthy donors and seven head and neck cancer patients. CD4(+) T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3(111-125) and MAGE-A3(161-175) epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4(+) T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor.